1 One-Minute NMR: Precious Samples using HPLC and Capillary NMR Natural Products Analysis The Easy Way with Microplate NMR Automation Easy Fast Efficient Economical Controllable Flexible Find the answers to… “Is this a known compound?” “What is the chemical structure? “How much do I have?” Copyright © 2006 by Protasis Corporation, All Rights Reserved

2 Challenges of Isolation and Elucidation of Natural Products Costly Sample Collection Challenging HPLC Separations Unknown Chemical Structures Purification-NMR Mismatches Complex NMR Spectra

3 X NMR Measurement Challenge What we care about What we don’t care about Mixture Pure Component LC Chromatogram LC Fraction

4 Analytical Scale HPLC Advantages Greater chromatographic resolution Smaller fractions fit in 96 or 384 well plates Less sample mass required Better detection options Reduced solvent use Perfect match for CapNMR probe

5 Purification-NMR Mismatches Temporal –HPLC fast, NMR slow Concentration –HPLC Fractions too dilute for good NMR Solvent –Protonated –Gradients –Salts H2O ACN

6 Sensible HPLC-NMR Vacuum Centrifuge Analytical HPLC DMSO-d6 One-Minute NMR High-Resolution Analytical Scale HPLC Simple Fraction Collection to Plates Pure Deuterated Solvents Concentrated Fractions for Maximum NMR Sensitivity Zero Sample Use

7 Protasis One Minute NMR Automation Sample Return Stacked Sample Drawers Wash Bays Microliter Precision Near Zero Dead Volume Easy Parameter Setup Solvent Conditioning Wellplate Web-based Software

8 One-Minute NMR Provides… Queue Management –Experiments, Structures, Fraction List Import Sample Loading –Vials and Plates, 5 & 10 microliter NMR Detection –Super-sensitive CapNMR Probe NMR Control –Adaptive Experiments Confirmation –ACD/Labs 1D + 2D Structure Verification Bonus: Quantitation –ERETIC Copyright © 2006 by Protasis Corporation, All Rights Reserved

9 MRM CapNMR TM Microcoil Probes 5-10 microliter flow cell = 8 – 15 microliter samples Best mass sensitivity Short acquisition times Concentration > 1mM works great (10 nanomoles in 10µL) Use CapNM R Copyright © 2006 by Protasis Corporation, All Rights Reserved Enhanced Mass Sensitivity Simple to Use, Easy to Shim! Teflon FEP Flowpaths

10 The Heart of MicroFlow NMR: The CapNMR Probe Sensitivity EconomySimplicity

11 Great Sensitivity, Easy Operation, Excellent Spectral Quality 600 MHz < 4 min 2 hr 6 hr Sensitivity EconomySimplicity

12 SOLV. A WASTE INJECT V2 LOOP SAMPLES NEEDLE SYRINGE High Pressure PUMP Copyright © 2006 by Protasis Corporation, All Rights Reserved CapNMR PROBE No Sample Loss With Sample Return Sample Return Option

13 One-Minute NMR Software Secure Log-In Individual sample entry and Molecule upload.ZIP/.CSV Sample lists and molecule upload Bar-code support Open SQL Server Custom Fields LDAP Login Web-Based Open Access Sample Queue Management Copyright © 2006 by Protasis Corporation, All Rights Reserved

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15 LEAP PAL Fast Low Volume Pickup Low Dead Volume Syringe-Only Technology Flexible Sample Storage Dilute / Reconstitute Sample Return Copyright © 2006 by Protasis Corporation, All Rights Reserved

16 Safe Solvent Management Vacuum Degasser –Smooth MicroFlows Helium Sparging / Blanketing –Protect Expensive Deuterated Solvents Helium NEEDLE V3 SAMPLES SYRINGE The Deuterated Solvent Conditioning Module mounts conveniently in the door of the Discovery Tower Vacuum Copyright © 2006 by Protasis Corporation, All Rights Reserved

17 Varian –SunTCP/IP Sockets Bruker –WindowsDrive Mapping –SGI / LinuxTCP/IP Sockets Others Easy instrument integration Spectrometer Interfaces Copyright © 2006 by Protasis Corporation, All Rights Reserved Router RSH(a.shimFile) RPAR(a.experiment, "all") SETCURDATA XCMD("gpro") RGA STOREPAR("NS", a.scans) STOREPAR("SW", a.sweepWidth) STOREPAR("o1p", a.centerOfSpectrum) exptime = CalcExpTime();

18 Use Case: Wellplate-Centric Workflow An Example from the University of Canterbury HPLC Fract Collect DAD ELSD Vacuum Centrifuge OMNMR Mass Spec MIM

19 Step 1: Chromatography Dionex HPLC Foxy Jr DAD ELSD u Dionex HPLC u ISCO Foxy Jr. Fraction Collector u 700 ug crude extract from culture of fungus Cladobotryum sp. u C18 column with d-ACN/D20 gradient with flow of 1mL/min u Eluant collected in 96-well conical bottom polypropylene plate u Receiving wells changed every 15 seconds (250uL/well) Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI (see March 2006 Gordon Conf. On Natural Products Presentations at

20 Step 2: Concentration u Evaporation using Jouan RC10-22 centrifugal vacuum evaporator u Plate enclosed with a piercible silicone/Teflon cover ~ 5 µg/well  6  g/well solvent u Trace amount injected into Mass Spec u Remainder available for additional analyses Mass Spec NMR

MHz 8  g in 6  L CD 3 OD PRESAT 1.5 min Recognisable features: 5 CH 3 groups, of which 1 methoxyl, 3 singlet CH 3 -C, 1 doublet CH 3 -C; 1 aldehyde Step 3: Analytical Measurement

22 only 1 hit Step 4: Database Search

23 Step 5: Optional – More Data Collection (in real time) 500 MHz (2 min) 30  g in 6  L CD 3 OD Unknown Peptide, MW= min TOCSY ? u Rapid 1D NMR enables real- time decision-making u Potentially novel compounds can be further analyzed using 2D experiments

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25 Wellplate-Centric Workflow An Example from Novatia LLC HPLC DAD ELSD Vacuum Centrifuge OMNMR Mass Spec Michrom HPLC or SepNMR

26 Buspirone Metabolites Metabolite Production –1.4 mg of Buspirone in 3ml with HLM –Reduce volume (vacuum centrifuge) to 400 ul Method –Column: Waters SymmetryShield RP8, 4.6X150 mm –Mobile Phases: A: 0.1% TFA in water, B:0.1% TFA in ACN –Method (1 mL/min) 0-13min at 4-63%B min 88%B 14-20min equilibrate –Collect fraction every 30 seconds (500 ul), or SepNMR

27 Step 1: Chromatography Step 2: Concentration Buspirone HLM Incubation Purified Buspirone Metabolite Sample: Incubate 700 ug buspirone with HLM Reduce volume (vac. cent.) to 400 ul Inject 80 ul on column Estimate 10 % MW 385 MW 401 Parent

28 Step 3: Analytical Measurement

29 Step 3: Analytical Measurement Step 4: Elucidation

30 TOCSY –CapNMR at 600 MHz –~ 5 ug buspirone metabolite –24 hours Step 5: Optional – More Data Collection (in real time)

31 One-Minute NMR is the practical solution for Natural Products, Metabolites, and Degradants. Isolate compounds with HPLC –Collect Fractions in Microplates Non-destructive screen with 1D- Proton NMR Identify / Elucidate interesting fractions with MS and 2D NMR

32 Help spread the word! Now NMR is easier and faster One-Minute NMR